MLL-AF4 leukemia in humans has long been known to have a unique and distinctive biology with a lymphoid or mixed lineage phenotype, high incidence in infants and a poor prognosis. The overall goal of this completive renewal is to expand our studies of the last 20 years that are designed to understand the unique molecular and cellular pathobiology of MLL-AF4 leukemia. Emphasis will be on our novel MII-AF4 knock in murine model developed recently in the Kersey laboratory as a result of technical advances over the past four years. Knock in models of MLL fusion gene leukemia have the advantage of expression of the fusion gene under control of the physiologic promoter and haploinsufficiency of MLL as in human leukemia To our knowledge this is the first MLL-AF4 model demonstrating an expansion of the lymphoid compartment in vitro and in vivo and eventual leukemia. This is significant because human MLL-AF4 leukemia develops within the lymphoid compartment. In contrast MLL-AF9 results in myeloid compartment expansion and myeloid leukemia;Human MLL-AF9 leukemia is generally myeloid. These results are consistent with an active and instructive (rather than a passive) role for the MLL partner gene in determination of the eventual leukemia phenotype. In the next grant period our specific aims are directed at detailed mechanistic studies interrogating the major hematopoietic progenitor and stem cell populations to understand the cellular basis for this active and instructive role of the MLL fusion partner (AF4 or AF9) in this selective expansion The MLL-AF4 mice to date have developed leukemia only after 5 months and somewhat unexpectedly have been myelomonocytic rather than lymphoid in type. In collaboration with Dr. David Largaespada we will work to develop a fully penetrant model of MLL-AF4 initiated lymphoid leukemia. In these studies we will evaluate cooperating mutations including FLT3 and mutations induced by END or a novel MuLV retrovirus. The possible role of haploinsufficiency of AF4 in the development of MLL-AF4 lymphoid leukemia will be evaluated. Our preliminary data indicate that 51 HOX A cluster genes play a significant role as targets for MLL fusion genes. In this grant period we will conduct detailed studies of 51 HOX A cluster and MEIS1 genes in the progenitor and stem cell populations in both MLL-AF4 and MLL-AF9 mice. Consideration will be given to HOXA cluster, MEIS1 or MLL fusion genes as potential therapeutic targets.